The overall goal of this project is to define specific steps in the calcium-dependent intracellular signaling events that modulate hydrochloric acid secretion by parietal cells in the gastric mucosa. Peptic ulcer disease is a widespread clinical problem that results from or is exacerbated by gastric acid secretion. A better understanding of the mechanisms by which calcium-dependent physiological agonists modulate gastric acid secretion will allow for improved treatment and prevention of this disease. Enhanced knowledge of the calcium signaling cascade has a much broader application as this pathway plays a major role in the control of secretion in a variety of cell types. This proposal will build upon previous observations in which a novel, low abundance calcium- dependent parietal cell phosphoprotein (now names calexin) was localized on 2-D gels, purified to homogeneity and partially sequenced. Amino acid sequence was used to clone the open reading frame using a degenerate primer PCR-based approach. The following specific aims will expand upon these observations by: 1) completing the cDNA sequencing of calexin and expressing the recombinant protein which can then be used to generate and screen for specific antibodies; 2) using northern, western and immunohistochemical techniques to characterize the tissue distribution of calexin; and 3) using the recombination protein and specific antibodies to characterize the dynamics of calexin phosphorylation in vitro and in vivo. The ultimate, long-term aim of this project is define the function of calexin in the calcium signaling pathway. Methodology to be used includes already established parietal cell isolation and primary culture techniques, cDNA cloning and sequencing, bacterial fusion expression, monoclonal antibody generation, northern analyses, protein phosphorylation and confocal immunofluorescent localization. Core facilities are available for monoclonal production, sequencing and confocal microscopy.